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Introduction Although there have been many advancesin green chemistry in the industrial andresearch fields, integration of theseconcepts into the teaching environment isstill in its infancy. This may be due to thelimited availability of educational materialsthat illustrate the methods, techniques, andprinciples of green chemistry. To addressthis problem, we developed a new greenorganic laboratory curriculum to teachfundamental chemical concepts andtechniques along with the tools andstrategies of green chemistry. 2–6 Integration of these goals into thelaboratory curriculum required thedevelopment of a broad collection of experiments that work well in thelaboratory, improve the safety of thelaboratory environment, and modernize thecurriculum through the introduction of state-of-the-art methods. Agreen organicchemistry laboratory textbook 2 and severalmanuscripts describe the criteria andprocess for greening experiments. 3,4,6 Here we describe a new laboratoryexercise that uses liquid CO 2 to extract D -limonene from citrus rind. This safe andconvenient procedure successfullyaddresses the diverse goals of greenchemistry experiment development bysimultaneously teaching practicaltechniques, fundamental chemicalconcepts, and green chemistry applications.In this experiment, the commonly taughtconcepts of natural product extraction andphase transitions are demonstrated througha novel procedure employing liquid CO 2 asa green solvent. The experiment introducesmodern green chemical approachesthrough discussions of industrial use of liquid CO 2 as a green replacement solvent( e.g. , dry cleaning) and supercritical fluidextraction (SFE) as an example of asuccessful commercial green process ( e.g. ,decaffeination of coffee). Replacement of traditional natural product extractionexperiments through incorporation of thisgreen chemistry research and technologypromotes an understanding of the currentpractice of green chemical methods. Thisexciting, convenient, and straightforwardprocedure offers an opportunity to teachcore organic chemistry concepts and skillsin the context of applicable greenchemistry.In this report, a short review of thebackground and current industrial uses of CO 2 and the relevance of this technologyto the teaching laboratory are described.The new laboratory procedure issummarized in the Experimental section. 7 In the Results and discussion section, boththe chemical and green lessons of thisprocess are discussed and extensions areproposed. Supercritical and liquid CO 2 At pressures above ambient, carbondioxide can exist in forms usable as asolvent ( i.e. as a liquid or supercriticalfluid). As shown in the phase diagram inFig. 1, CO 2 is a liquid under relativelymild temperatures and pressures, in theranges of 2 56.6 to 31.0 °C and 5.2 to 73.8bar. Supercritical carbon dioxide (scCO 2 )is produced at temperatures higher than thecritical temperature (31.0 °C) and betweenthe critical pressure (73.8 bar) andextremely high pressures (approximately10 4 bar). Supercritical fluids have nodistinct liquid or vapor phase but retainproperties of each. ScCO 2 is especiallybeneficial when used as a solvent inselective extraction processes. The gas-likeproperties, such as very low surfacetension and viscosity, allow the solvent topenetrate into the substrate, while theliquid-like properties solubilize compoundsand remove them from the substrate. Smallchanges in pressure or temperature alterthe bulk density of the fluid leading toincreased or decreased solubility of variouscompounds. In this way, the use of supercritical fluids can allow for control of separations of materials. Throughmanipulation of temperature and pressureconditions within accessible ranges, boththe phase and properties of CO 2 can beeasily controlled.During the past two decades, technicaladvances have been made in the industrialuse of supercritical and liquid carbondioxide in place of organic solvents. 8–10 CO 2 is useful as a green alternative solventbecause it provides environmental andsafety advantages: it is nonflammable,relatively nontoxic, readily available, andenvironmentally benign. Processing withCO 2 also poses minimal hazard in theevent of unintentional release or residualsolvent in the product. Although CO 2 is agreenhouse gas, when used as a solvent itis captured and employed, not generated,resulting in no net environmental harm.Additionally, a closed loop system can beused to compress the gas in order to usethe supercritical fluid or liquid inprocessing, depressurize the solvent forseparation of dissolved compounds, andrecompress the CO 2 to begin the cycleagain. CO 2 extraction processes can alsobe run at relatively constant pressure whenliquid–liquid extraction against water isused for product recovery. These loopsystems allow for easy recovery andrecycling of the pure solvent. This journal is © The Royal Society of Chemistry 2004 Green Chem., 2004 , 6 , 355–358 355 Green chemical processing in the teaching laboratory: a convenientliquid CO 2 extraction of natural products† Lallie C. McKenzie, a John E. Thompson, b Randy Sullivan c and James E. Hutchison* a a Department of Chemistry and the Material Science Institute, University of Oregon, OR 97403, USA. a E-mail:
[email protected] b Department of Chemistry, Lane Community College, OR 97405, USA c Department of Chemistry, University of Oregon, OR 97403, USA Aunique liquid CO 2 extraction laboratory developed for a greener organic teaching lab curriculum provides aneffective, inexpensive, and convenient procedure for teaching natural products extraction concepts and techniquesusing modern green extraction technology. The procedure is appropriate for the teaching lab, does not require anyspecial equipment, and allows the students to see the phase change and extraction as they occur. Students learnextraction and spectroscopic analysis skills, are exposed to a dramatic visual example of phase change, and areintroduced to commercially successful green chemical processing with CO 2 . D O I : 1 0 . 1 0 3 9 / b 4 0 5 8 1 0 k C G www .r s c . or g / gr e en ch em FOCUS †Electronic supplementary information (ESI)available: experimental procedure (includinginstructors’notes, student handout, anddemonstration procedure) and movie clip of extraction process. See http://www.rsc.org/ suppdata/gc/b4/b405810k/ Green Chem., 2004 , 6 , 355–358 356 Applications of CO 2 as an alternativesolvent Large-scale CO 2 processing has hadcommercial success in many separationand extraction processes. 8,9 The tunablesolubility properties, low toxicity, and easeof removal of CO 2 have led to wellestablished scCO 2 technology for theextraction of various food products,including essential oils and hops, and forthe decaffeination of coffee and tea. Themild conditions necessary for extractionand absence of residual solvent result insuperior products and have motivated anindustrial shift to CO 2 from hazardoussolvent extractions or steam distillations.The oil products from scCO 2 extractionprocesses are of higher purity and containno thermal degradation products. Theability to influence solubility of compounds through variations intemperature and pressure has resulted inenhanced extraction of desired compoundsfrom natural products and in the ability toenrich oils during post-extraction treatmentwith CO 2 . 11,12 ScCO 2 has also been usedin other processes including analyticalextractions and chromatography, metaldegreasing, and textile dyeing.Acommitment to replacing hazardoussolvents and improving environmentalfootprints has led to many new greenmethods of materials synthesis andprocessing with CO 2 . 8,9,13 Carbon dioxideis relatively inert, is resistant to oxidation,cannot serve as a chain transfer agent, andprovides for tunable miscibility. Thesechemical advantages have led to anincreasing number of industrial-scalereactions which use CO 2 . Carbon dioxidehas been employed in the synthesis of polymers such as DuPont™Teflon®fluoropolymer resins and for commercial-scale hydrogenation and oxidationreactions. 8 The UNICARB®VOCReduction Process uses scCO 2 as thecarrier and atomizing agent for sprayingpaints and coatings. 9 The variablesolubility permits the use of CO 2 in theformation of micron-sized particles,technology which has been employed inthe synthesis of inhalable medications. 14 Inaddition to these demonstrated uses forCO 2 , current research is also investigatingits applicability in the microelectronicsindustry, in catalysis reactions, and as asimultaneous reaction medium and rawmaterial. 13 Although most industrial applicationsuse supercritical CO 2 , liquid phase carbondioxide has also proven effective. Thewide range of lower temperatures andpressures offers flexibility in the design of processes using liquid CO 2 . The mildconditions prevent degradation of products.As with scCO 2 , ideal density, viscosity,and surface tension can be obtainedthrough manipulation of pressure andtemperature. Liquid CO 2 has beenemployed extensively as an industrialsolvent for the extraction of essential oils 15 and in new greener methods of drycleaning. 8,9 To date, a convenient liquid CO 2 extraction method using standardlaboratory materials has not beenreported. The laboratory experiencedescribed here brings the liquid CO 2 extraction process into the teaching orresearch laboratory in an inexpensive,effective, and accessible manner. It offersan opportunity for students to learnextraction techniques, observe strikingphase changes, and appreciate the benefitsof using greener chemical methods. Thiscarbon dioxide extraction procedureprovides a convenient drop-in replacementfor currently used natural product steamdistillation or solvent extractionlaboratories. Experimental Dry ice sublimes at atmospheric pressureand temperatures above 2 78 °C. If theCO 2 is sealed in a vessel duringsublimation, the internal pressure in thevessel increases. After the temperature andpressure have increased sufficiently, liquidcarbon dioxide forms. Due to thisaccessible phase change, carbon dioxidecan be used for bench top extractionprocesses. In this experiment (see Fig. 2and ESI†), approximately two and a half grams of grated orange peel and a wire andfilter paper or metal screen solid trap areplaced in a 15 mLpolypropylenecentrifuge tube with plug seal cap (Corningcatalog #430052). 16 The centrifuge tube iscompletely filled with crushed dry ice,capped tightly, and dropped (tapered enddown) into a plastic cylinder orpolycarbonate bottle which is half-filledwith warm (40–50 °C) tap water.As pressure builds in the tube, gasescapes slowly through the threading of thecap. 17 After approximately fifteen seconds,the solid begins to melt, and liquid CO 2 appears in the tube. Solid, liquid, and gasphases are visible in the tube for a shortperiod of time. 18 The liquid boils, and gasescapes for almost three minutes. Duringthis time, the liquid CO 2 moves throughthe solid, extracts the oil from the orangerind, and collects in the bottom of the tube.The solid trap successfully prevents theorange rind from moving into the tip of thetube during extraction because the wirecoils are supported by the sides of thecentrifuge tube at the point where itnarrows. After the extraction solventcompletely evaporates, isolated productremains in the tip of the tube. Once theliquid has stopped bubbling and gas is nolonger escaping, the centrifuge tube isremoved from the cylinder with tweezers,and the extraction process is repeated byrefilling the tube containing the orangerind and solid trap with dry ice, recappingit, and replacing it in the cylinder. Two orthree extraction cycles result in isolation of approximately 0.1 mLof pale yellow oil.Typical yields are comparable to organicsolvent extraction or cold pressing (1–2%recovery, based upon initial mass of rindused during extraction). 19 The extractedproduct is predominantly D -limonene by 1 H NMR and IR analysis and 97% D -limonene as indicated by GC-MS. Results and discussion The inspiration for this laboratory camefrom a desire to develop a reliable,convenient, inexpensive, and safe approachto CO 2 extraction that could be performedeasily in any teaching laboratory. Asecondary goal was to allow students tovisually observe the phase changes of CO 2 .Several liquid and supercritical CO 2 Fig. 1 The temperature–pressure diagram for carbon dioxide clearly indicates the pressures andtemperatures for the phase transformations, triple point, and critical point. Adapted and used withpermission from ChemicaLogic Corporation. 1 Green Chem., 2004 , 6 , 355–358 357 procedures have been developedpreviously, but these methods show thephase change for a brief time and do notallow for extraction, limit visualization of the phase transitions, or require expensiveextraction equipment. 20–22 We sought tomake carbon dioxide extraction of naturalproducts widely accessible and lengthenthe observation period of phase changes,while maintaining a high degree of safety.The following sections detail the coreorganic laboratory concepts and skills aswell as the green lessons taught throughthis new approach. Although the scope of this report is limited to a summary of thelaboratory experience, more specificinformation and detailed instructions areincluded in the ESI.† Natural products extraction andspectroscopy Organic laboratory courses often include anatural product extraction in order tointroduce solid/liquid extraction methodsand the chemistry of the terpenecompounds. The most commonly usedmethods are those of steam distillation andorganic solvent extraction. In the proceduredescribed herein, the techniques of liquid/solid extraction are taught throughrepeated extractions of orange peel withcarbon dioxide (see Scheme 1). Theintroduction of CO 2 extraction into thecurriculum allows students to use a greensolvent and to compare and contrastseparation techniques. The practical andreliable procedure provides reproducibleresults and isolable yields of pure productwhich are comparable to those of otherextraction methods. The volume of productcollected is large enough for analysis byspectroscopic methods, and the use of 1 HNMR and IR spectroscopy providesopportunities for detailed analysis of complex spectra. Gas chromatography-mass spectroscopy can be used to comparethe purity of samples generated bydifferent extraction methods or to evaluatethe composition of oils from differentcitrus fruits. Phase transitions Changes in physical states of compounds,the effects of temperature and pressure onthese states, and their representation withphase diagrams are discussed often inchemistry courses. The opportunity tointroduce these concepts in an easy andvisual manner is a challenge addressed byfew laboratory materials. 20,21 Students arefamiliar with the sublimation of CO 2 andwith the use of CO 2 in fire extinguishersand fountain drinks. Carbon dioxideprovides accessible phase changesrequiring low temperatures and relativelylow pressures. In particular, the liquid stateis accessible through slight increases inpressure. During this laboratoryexperiment, the solid dry ice melts quicklywhen appropriate pressure and temperatureconditions are reached. For at least oneminute, solid, liquid and gas phases arevisible. The transparent plastic extractionvessels and containment cylinders used inthis procedure allow the students toobserve these phase changes safely andeasily. Green messages In this laboratory, there are opportunitiesfor discussions of many green chemistryprinciples. 23 Primarily, the focus is onprevention of waste and using safersolvents. Students are encouraged toconsider the effects of solvent choice andextraction method on the extractionprocess and product. Using CO 2 as asolvent presents no risk to human health orthe environment, and the extraction is aseffective as extraction with othercommonly used solvents. Both solventextraction and steam distillation proceduresproduce significant amounts of solventwaste for very little product, but there is nosolvent waste when carbon dioxide is used.The product of this extraction exhibitshigher purity due to the absence of solventresidue. Students also are exposed to agreen chemical process which has beenwidely incorporated into industrialpractice. This view of green chemistry asan active and applicable set of principlesimproves the students’perception of chemistry and provides beneficialpreparation for students who may becomechemists in industrial or academic settings. Extensions Due to the pressure limitations of thesimple equipment used in this experiment,extractions with this procedure are limitedto those that can be performed in liquidCO 2 . Specially-designed equipment isrequired to extend to supercritical fluidextractions (SFE). Teaching laboratoryinstructors must balance the need for aninexpensive and convenient procedure withthe opportunity to demonstrate theversatility of CO 2 extraction. While lab-scale supercritical fluid extractors areavailable, they are expensive. If financiallyfeasible, introducing SFE technology intothe teaching laboratory through the use of supercritical fluid equipment wouldprovide opportunities for extraction of awide variety of compounds and improvedquantitative analysis.Extensions of this laboratory exerciseinclude further exploration of theprinciples and practice of green chemistry.This procedure can be used to helpstudents improve their understanding of theconcepts of cleaner processing through theevaluation of yields, purity, wastegenerated, and energy costs of differentextraction methods. 2 Through readingscientific literature, students can explorethe current trends of industrial processingwith CO 2 . Discussion of the benefits anddifficulties of using CO 2 as a green solventcan provide students with an awareness of the challenges involved in solventreplacement.Although supercritical and liquid CO 2 have been used to extract natural productsfrom coffee, hops, and many fruits, Fig. 2 Illustration of the liquid CO 2 extraction procedure. Asolid trap is constructed by (A)bending copper wire into coils and a handle, (B) placing filter paper or metal screen between thewire coils, and (C) placing the solid trap in a centrifuge tube. For extraction, (D) grated orange peelis placed in the tube, and (E) the tube is filled with crushed dry ice and sealed with a cap. (F) Theprepared centrifuge tube is placed in the water in the graduated cylinder, and the liquefaction andextraction occur over the following three minutes. Scheme 1 Extraction of D -limonene fromgrated orange rind using liquid CO 2 as a greenextraction solvent. The product is 97% D -limonene and has no solvent residue. Green Chem., 2004 , 6 , 355–358 358 flowers, and spices, 15 this laboratoryprocedure has only successfully extractednatural products from citrus rinds.Modification of the extraction conditionsto include a larger amount of substrate arerequired to allow for extraction of materials of lower density, and grinding orother processing may be necessary whenintroducing large solid matrices.This lab is readily adapted as aclassroom demonstration. Visibility isimproved, and safety is enhanced byplacing the centrifuge tube in water in alarge polycarbonate cylinder. Using thisapparatus, students may view thedemonstration at closer range. Thedemonstration also may be projected usinga video camera and projector. 24 Conclusions The liquid CO 2 extraction laboratorydescribed herein incorporates moderngreen chemistry into the organic teachingenvironment in a visible and excitingmanner. Equally important, this procedurefits the constraints of the teachinglaboratory, including those of time, safety,effectiveness, affordability, andconvenience. The one to two hourprocedure, including all preparation,extraction, and analysis, can be performedon its own or conducted concurrently withanother experiment. In this laboratoryexercise, all chemical hazards of traditionalextraction procedures have been removed,and laboratory conditions provide for safeviewing of the phase changes andextraction. All required materials arereadily available and inexpensive. Unlikesteam distillation or organic solventextraction procedures, there is no wastedisposal cost with CO 2 extraction. 25 Comparisons of product recovery andpurity, generation of waste, and risk tostudents and the environment indicate thatthis procedure provides the greenestnatural product extraction laboratorycurrently available. 26 The convenient, rapidliquid CO 2 natural product extraction isbased on the foundation of commerciallysuccessful green chemical processing.Through the application of principles andstrategies of green chemistry andinstruction in practical techniques andconcepts, this drop-in replacement naturalproduct extraction laboratory exerciseeasily teaches fundamental chemicallessons and successfully incorporates greenchemistry into the teaching laboratory. Acknowledgements This work was supported by the Universityof Oregon, the National ScienceFoundation (CHE-9702726 and DUE-0088986), and the American ChemicalSociety. We thank Gary Nolan for hisassistance with GC/MS data collection,and the students enrolled in OrganicLaboratory at Lane Community Collegefor their assistance in optimizing andtesting this experiment. J.E.H. is an AlfredP. Sloan Research Fellow and a CamilleDreyfus Teacher-Scholar. References 1ChemicaLogic Corporation.http://www.chemicalogic.com/download/co2_phase_diagram.pdf (accessed Feb 2004).2K. M. Doxsee and J. E. Hutchison, GreenOrganic Chemistry: Strategies, Tools, and Laboratory Experiments , Brooks/Cole,Pacific Grove, CA, 2004.3S. M. Reed and J. E. Hutchison, J. Chem. Educ. , 2000, 77 , 1627–1629.4M. G. Warner, G. L. Succaw and J. E.Hutchison, Green Chem. , 2001, 3 , 267–270.5L. C. McKenzie, L. M. Huffman, K. E.Parent, J. E. Hutchison and J. E. Thompson, J. Chem. Educ. , 2004, 81 , 545–548.6L. C. McKenzie, L. M. Huffman and J. E.Hutchison, The Evolution of a GreenChemistry Laboratory Experiment: GreenerBrominations of Stilbene, J. Chem. Educ. , inpress.7Detailed notes for instructors, spectral data,student handout, and demonstrationprocedure are available in the electronicsupplementary information (ESI) of this journal. See http://www.rsc.org/suppdata/ gc/b4/b405810k/.8E. J. Beckman, Environ. Sci. Technol. , 2002, 36 , 347A–353A.9E. J. Beckman, Ind. Eng. Chem. Res. , 2003, 42 , 1598–1602.10The two reviews cited here provide detailedbackground information on currentsustainable materials processing with CO 2 . Alist of websites which provide informationabout specific CO 2 technologies is includedin the instructors’notes in the ESI.11F. Benvenuti and F. Gironi, J. Chem. Eng. Data , 2001, 46 , 795–799.12A. Chafer, A. Berna, J. B. Monton and A.Mulet, J. Chem. Eng. Data , 2001, 46 ,1145–1148.13N. Tanchoux and W. Leitner, in Handbook of Green Chemistry and Technology , eds. J.Clark and D. Macquarrie, BlackwellScience, Oxford, 2002, pp. 482–501.14Thar Technologies, Supercritical FluidParticle Formation Home Page.http://www.thartech.com/systems/particle/ (accessed Feb 2004).15M. Mukhopadhyay, Natural Extracts UsingSupercritical Carbon Dioxide ; CRC Press,Washington, DC, 2000.16This procedure has only been successfullytested with centrifuge tubes of this brand andpart number. The caps of larger Corningtubes (50 mL) did not withstand thepressure. The tubes must withstandtemperatures from 2 78 to 50 °C andpressures from 1 to at least 6 atm. For safetyreasons, the ability to withstand higherpressures is desired.17Under experimental conditions, carbondioxide gas leaks from the centrifuge tube atan average rate of 2.5 g min 2 1 .18The simple apparatus used in this experimentdoes not allow for accurate determination of temperature and pressure conditions underwhich the extraction is occurring. Althoughthe system is not at equilibrium, the phasediagram and physical observations can beused to estimate the conditions in the tube.Observation of both the gas/liquid andsolid/liquid interfaces indicates that locallythe temperature is between the temperatureat the triple point ( 2 56.6 °C) and the criticaltemperature (31.1 °C). Formation of ice onthe surface of the tube further brackets thetemperature to between 2 56.6 and 0 °C. Thepressure is passively regulated by the leakingfrom the tube and, due to the liquefaction, isassumed to be above the pressure at thetriple point (5.2 bar) but below the pressurethat would induce tube rupture. Theseobservations indicate that conditionsapproach those of the triple point.19D. C. Smith, S. Forland, E. Bachanos, M.Matejka and V. Barrett, Chem. Educ. , 2001, 6 , 28–31.20V. T. Lieu, J. Chem. Educ. , 1996, 73 ,837.21 Introduction to Green Chemistry: Instructional Activities for IntroductoryChemistry , eds. M. A. Ryan and M.Tinnesand, American Chemical Society,Washington, DC, 2002.22N. H. Snow, M. Dunn and S. Patel, J. Chem. Educ. , 1997, 74 , 1108–1111.23P. T. Anastas and J. C. Warner, GreenChemistry: Theory and Practice , OxfordUniversity Press, New York, 1998.24Detailed demonstration procedures areincluded in the ESI.25Cost per student is approximately $1.00 forlaboratory materials including centrifugetubes, dry ice, and citrus fruit. Plasticcontainment cylinders can be purchased orconstructed for $2–3 each. One cylinder perstudent per lab period will be required.Further details are provided in the ESI.26Product recovery ranges from 1–2% basedon initial mass of rind used. GC-MS data of student-performed steam distillation, pentaneextraction, and carbon dioxide extraction areavailable in the ESI.
